Immunoassays have been used for decades as a means to assay for the qualitative and quantitative presence of antigens or antibodies in a sample. Among the most common immunoassay techniques include a solid phase matrix to which either an antigen or antibody is bound. While numerous methods for attaching the antigen or antibody to the solid phase are known and several widely used, the attachment technique remains an important step in the preparation of an immunoassay. Indeed, the immobilization of an antibody or antigen to the solid phase is usually one of the first steps in preparing an immunoassay.
Swan, et al. (BSI Corporation), U.S. Pat. No. 5,414,075, disclose chemically coupling a target molecule, such as phospholipids, to a plastic support, such as polystyrene, using a multi-functional chemical coupling agent.
Sharma, U.S. Pat. No. 4,360,358, discloses the formation of an immunologically active solid phase by incorporating a low molecular reagent, such as a hapten, into a material which forms a solid polymer. This polymer is itself coated on a solid phase. The base solid phase may be polystyrene. The material forming a solid polymer includes a number of gels and the like.
Sterhan et al. (Biostar Medical Product, Inc.), World Patent 90/10227, disclose the absorption of cardiolipin, phospholipids and other materials on a solid support, such as a plastic well plate, which was previously coated with methylated bovine serum albumin.
Shah et al. (Baxter Diagnostics Inc.), World Patent 91/10138, disclose either the passive absorption or the chemical coupling of cardiolipin, phosphatidylcholine and/or cholesterol to polystyrene plates for the purposes of an ELISA (enzyme-linked immunosorbent assay). Note that all of the coating and coupling occurs after the plastic plate has been formed.
Matsuura, et al. (Yamasa Shoyu Kabushiki Kaisha), U.S. Pat. No. 5,506,110, disclose binding various phospholipids on a polystyrene well plate for the purposes of an ELISA. The antigens are passively absorbed on the solid phase. Note the assay's ability to distinguish between various antiphospholipid antibodies.
Lostia, et al. (Snam Progetti S.p.A.), U.S. Pat. No. 4,031,201, disclose the preparation of fibers incorporating antibodies or antigens. These fibers are used for a number of purposes including as a solid phase in various immunoassays. The active substance, which may be a hapten, is mixed with a polymer and the mixture is then spun through a coagulation bath to produce the solid phase in the form of fibers. Note that the polymer may be polystyrene. The fibers are porous and contain microcavities.
Peters, Jr. et al. (SmithKline Diagnostics, Inc.), U.S. Pat. No. 5,013,669, disclose an immunoassay wherein the antigen or hapten is chemically coupled to a polymer which forms a solid material. This material is coated on another solid support. The polymer is reversibly water-soluble and is chemically bound to the antigen/hapten.
Sutton, (Eastman Kodak Company), U.S. Pat. No. 5,234,841, discloses the coating of a solid phase, such as polystyrene with an antigen/hapten for use in an immunoassay. The biologically active material is dissolved in a solvent and then coated on the solid phase.
Bonacker, et al. (Behringwerke Aktiengesellschaft), U.S. Pat. No. 4,118,349, disclose immunoassays wherein the solid phase immobilized antibody or antigen is chemically bound to a polystyrene solid phase. The antibody or antigen is chemically coupled to the polystyrene carrier through a chemical coupling compound.
Yabusaki (Hana Biologies, Inc.), U.S. Pat. No. 4,459,362, discloses an immunoassay for antibodies to various phospholipids using phospholipids in suspension.
Hartdegen, et al. (W. R. Grace & Co.), U.S. Pat. No. 4,195,127, disclose immobilizing proteins, which include antibodies or antigens, in a polyurethane foam product. The antibody or antigen is mixed with a monomer or prepolymer and reacted thereto. The chemical conjugate of antigen and prepolymer is then polymerized to form the polyurethane foam. This solid phase can then be used for a number of uses. The antibody or antigen is chemically coupled to the polymer molecule.
Nowinski, et al. (Genetic Systems Corporation), U.S. Pat. Nos. 4,609,707 and 4,752,638, disclose the formation of a polymer-antibody or polymer-antigen solid material by chemically reacting the antibody or antigen to a monomer directly or indirectly to form a monomer/antibody or antigen conjugate, followed by polymerizing of the monomer. The material may then be used as a solid phase in immunoassays. The solid phase may take any form.
Johnson, et al. (Miles Inc.), U.S. Pat. No. 4,822,747, disclose immobilizing a hapten reagent on a solid phase and using it in an immunoassay. The reference teaches that the hapten is to be chemically coupled to reactive moieties on the outer surface of the polymer. Interesting, Johnson, et al. emphasizes the need to chemically bind the hapten to the solid phase as non-specifically bound haptens may be washed or slowly leached away from the solid phase.
Walter, (Miles Laboratories, Inc.), U.S. Pat. No. 4,390,343, discloses dipstick-type analytical elements where the antibody or antigen/hapten are incorporated into a gel, such as agarose, gelatin or PVP.
Immunoassays for detecting syphilis have been in widespread use for decades. Every unit of blood and patients suspected of having any sexually transmitted disease are routinely screened for syphilis by immunoassay. The techniques for screening blood for antibodies to syphilis have included VDRL (Venereal Disease Research Laboratory), RPR (rapid plasma regain), complement fixation, treponemal immobilization/adherence, FTA (fluorescent treponemal antibody) ELISA and possibly a number of other immunoassay formats also. The earliest immunoassay for phospholipids (PL) is the Wassermann reaction (ca. 1905) which is a complement fixation assay.
All immunoassay methods are dependant on antibody binding to the antigen. The antigen for syphilis serology has historically been an alcohol extract from beef heart mixed with cholesterol. The antigen (cardiolipin) was typically adsorbed onto carbon particles as a solid phase. While the antigen is not perfect, it has demonstrated its effectiveness at protecting the blood supply.
A number of other diseases also have been associated with or identified by detecting antibodies to the same PL antigens or to phospholipid binding proteins. Examples include patients with systemic lupus erythematosus (SLE) and a subset of patients identified as having anti-phospholipid syndrome. Clinical findings include recurrent venous thrombosis, recurrent arterial thrombosis, recurrent spontaneous abortion, thrombocytopenia, chorea, epilepsy, livedo and idiopathic pulmonary hypertension. Other rheumatological and collagenous diseases also present as characteristic antibodies to PL in the patient's serum. In the field of human organ transplantation, primary non-function of the organ also may appear associated with the presence of anti-phospholipid antibody (aPA). Wagenknecht et al, Human Immunology, 49, p. 27 (1996). Accordingly, there is a great need and numerous applications for a standardized immunoassay for aPA.
ELISAs has been in use for about 25 years to detect small amounts of antigenic substances. Today, many ELISA systems use plastic (polystyrene) 96-well plates (Microtiter plates) which have been adapted and/or modified to provide optimal binding of the antigenic substance to which antibodies have been produced. Beginning in the 1980's, the ELISA was selected for use for the detection of antibodies to antigens composed of PL and/or PL-binding plasma proteins.
The term "anti-phospholipid antibody" (aPA) refers to the conventional usage of that term in which many antibodies to PL are actually antibodies to plasma proteins which bind to phospholipids. Nonetheless, PL are considered an antigen. While not wishing to be bound by any theory, it is believed that phospholipid binding proteins do not bind aPA in the absence of another component such as PL unless the plastic containers are specifically treated such as irradiated plastic surfaces which increase the binding of certain phospholipid binding proteins. See McIntyre et al, American Journal of Reproductive Immunology, 37: p. 101-110 (1997). While the term "anti-phospholipid antibody" may be confusing, it is well understood in the art.
Since its inception, immunoassays used for detection of anti-phospholipid antibody (aPA) in patient blood have been fraught with problems relating to reproducibility, sensitivity and quantification. Interlaboratory comparisons of quantification of aPA are particularly in disagreement. See Wagenknecht et al, Clinical Immunology Newsletter, 15(2/3):28-38 (1995) for a review article on the subject. A major problem with all ELISA systems and especially with the aPA ELISA involves the important first two steps in the assay; 1. "coating" the antigenic material to the plastic surface of the microtiter plate wells and 2. "blocking" with a proteinaceous substance, often bovine serum albumin. The blocking step may remove PL from the plate because serum albumin and other proteins can bind to PL and thus they compete with the PL coated plastic plate for PL binding. Additionally, it is difficult to control the blocking step to prevent this loss.
In the aPA ELISA, PL such as cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyldiglycerol, phosphatidylinositol, phosphatidylcholine and phosphatidic acid are used to coat the wells of the plates. This coating step often is done in the presence of organic solvents, such as chloroform, which are desirable for their ability to keep lipids in solution. Organic solvents are undesirable, however, because they can etch plastic and thus cause unwanted light refraction when the plate contents are measured for changes in optical density, i.e., color. Also, it has been reported that up to 64% of the phospholipid antigen was removed from the plastic surface during the course of the assay. See Smolarsky, Journal of Immunological Methods, 38: p. 85-93 (1980) Organic solvents are not acceptable for certain proteinaceous substances which may be denatured and inactivate their binding properties.
After the PL is applied to the wells, a second problem may be oxidation of the PL containing fatty acids. The oxidation of unsaturated fatty acids causes them to become rancid and/or to cross link. Crosslinking by oxidation is the basic principle occurring in the "drying" of paints and varnishes. Oxidation has been shown to alter the antigenic properties of some PL. To avoid this problem, aPA ELISA plates often are dried under nitrogen or stored in sealed containers.
A third problem is the loss of the coated antigen due to the blocking agent and numerous wash steps required during the course of the ELISA. Historically, multiple steps in aPA ELISA plate preparation generate other problems and expenses such as the cost of labor intensive setup procedures. Due to the lack of a consensus about PL coating of the plate among laboratories who perform these assays, poor intra- and interlaboratory reproducibility has been a consistent problem.
Similar problems exist with the immobilization of other antigens and antibodies to plastic surfaces. Other binding assays and other surfaces have similar problems. Regardless of the antigen (or antibody), protein, phospholipid, carbohydrate (such as blood group antigens), or nucleic acid, all have certain inherent problems when immobilizing on a plastic surface, not the least of which is retaining their binding properties.
Many untreated plastics are hydrophobic. If a binding agent being immobilized has a hydrophobic portion, this portion will tend to adhere to the plastic surface if an aqueous carrier solute is used. Depending on the solute and surfaces being used, the complementary portion of the binding agent will tend to adhere to the plastic surface. In immunoassays, the antibody-antigen binding occurs on only a specific portion of each molecule. The same is true for other receptor/ligand binding assays. If this portion is obscured by the plastic surface, binding will be inhibited. This can lead to irreproducible results, lower than true concentrations and even false negatives.
Similar problems exist with the immobilization of any ligand or receptor in a binding assay, affinity binding or chromatography system, enzyme immobilization and for the coating of medical implants to make them more acceptable to the recipient.
The presence of or levels of antibody, antigen, or ligand in a biological sample is indicative of various conditions and is diagnostically important. For example, antibody to PL in the serum may be indicative of whether an infection remains or an autoimmune disease state is likely. Past exposure to the antigen, such as with a treated syphilis infection, without active current infection may also be detected but the antibody titer will eventually be lower as time passes, particularly IgM and IgA titers. Likewise, high levels of aPA correlate with further occurrence of thrombosis in patients with SLE. To quantify the level of antibody in blood, one must have a reproducible standard assay since there is a correlation between the number of antigen molecules on the solid support and the number of bound antibody molecules resulting from sample application.